Subcellular localization and secretion of factor V from human platelets.

Chesney, Carolyn M.; Pifer, D. David; Colman, Robert W.

doi:10.1073/pnas.78.8.5180

Cited by 70 publications

(35 citation statements)

References 30 publications

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“…Thus far, megakaryocytes have been shown to be able to synthesize Factor VIII-related antigen (19), platelet Factor 4 (24), fibrinogen (25), and actin (22). The observation that guinea pig megakaryocytes can synthesize Factor V suggests that granule development occurs early in megakaryocyte development since Factor V's, at least in the case of platelets, are located in a-granules (7).…”

Section: Resultsmentioning

confidence: 99%

“…However, Factor V may also arise from an intracellular location in platelets since human platelets separated by gel filtration do not have significant Factor V activity but develop coagulant activity upon repeated freezing and thawing (6). Studies from this laboratory show that both human (7) and bovine (8) platelets contain Factor V in the a-granules where it can be released by collagen. Thrombin can also release Factor V from human platelets (9), where it would be in the form of Factor Va and thus would exhibit high affinity binding to the surface membrane.…”

Section: Introductionmentioning

confidence: 92%

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Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Chiu¹,

Schick²,

Colman³

1985

J. Clin. Invest.

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Although platelets contain Factor V, localized primarily in the a-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chin, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098±0.018 gg/105 cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234±0.180 gig/108 platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0±3.0 gig/ml) than that of Factor V antigen in human plasma (11.1±0.4 gig/ ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01±1.09 gg/108 platelets) than do guinea pig platelets. The Factor V coagulant activities in the guinea pig were 2.85±030 U/ml plasma, 0.022±0.012 U/108 platelets, and 0.032±0.03 U/105 megakaryocytes, compared with human values of 0.98±0.02 U/ml plasma and 0.124±0.064 U/1O8 platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with V35Sjmethionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Chiu¹,

Schick²,

Colman³

1985

J. Clin. Invest.

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“…Substantial binding of factor Xa (in the absence of added factor Va) was observed on thrombin-stimulated adherent platelets (Fig 5). Because factor Va is released from the platelets on activation, 69 -70 the factor Xa binding results suggested that the binding of factor Xa to thrombin-stimulated adherent platelets was at least partially dependent on plateletreleased factor Va. However, the binding of factor Xa did not saturate on thrombin-stimulated adherent platelets even at a solution concentration of 400 nmol/L, which is approximately fourfold greater than the normal plasma concentration of factor X.…”

mentioning

confidence: 87%

“…aBFV-4 allowed definitive assessment of specific factor Va/factor Xa interactions on the platelet surface. The antibody also allowed for discrimination between added bovine factor Va and platelet-released human factor Va. (Human platelets contain factor V in their a-granules, 69 and they release the cofactor on activation.…”

Section: Discussionmentioning

confidence: 99%

The assembly of the prothrombinase complex on adherent platelets.

Swords

Mann

1993

Arterioscler Thromb

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Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated K a of 58 nmoI/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no K d value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated K A for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa. (Arterioscler Thromb.

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“…Results from our laboratory indicate that prothrombin activation occurs on the platelet surface (200-300 sites/cell) (1)(2)(3)(4)(5)(6). In addition to being present in plasma, coagulation Factor V is present in platelets localized in the a-granules (7)(8)(9). Platelet Factor V can be released by a variety of agents that stimulate platelet secretion (6,8,9) and once activated to Factor Va, can serve to promote Factor Xa binding to platelets (6).…”

Section: Introductionmentioning

confidence: 99%

Activation of Coagulation Factor V by a Platelet Protease

Kane¹,

Mruk²,

Majerus³

1982

J. Clin. Invest.

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A B S T R A C T Factor V must be converted to Factor Va in order to bind to a high affinity platelet surface site and participate in prothrombin activation. Osterud et al. (10) presented data that suggested that human platelets contain an activated form of Factor V and a Factor V activator. We find that the Factor V released when platelets are disrupted by freezing and thawing or sonication is activated 3-to 10-fold by thrombin as determined by coagulation assay and is therefore stored as the relatively inactive procofactor rather than in the active form Factor Va.We incubated purified Factor V, which had a specific activity of 140±30 U/mg, with Factor V-deficient frozen and thawed platelets (10' platelets/ml) obtained from a patient with Factor V deficiency. The specific activity of the Factor V increased to a maximum of 740±240 U/mg (mean±SD of three experiments). When this partially activated Factor V was incubated with thrombin its specific activity increased further to 1,440±280 U/mg, which is similar to the activity of Factor V activated with thrombin alone (1,540±60 U/mg).The platelet Factor V activator is not inhibited by dansyl arginine-4-ethylpiperidine amide, 93 iM, indicating that it is not thrombin. When thrombin-stimulated platelets, to which dansyl arginine-4-ethylpiperidine amide had been added to inhibit the further action of thrombin, were incubated with '25-labeled Factor V, there was no detectable proteolysis of the Factor V molecule. Our failure to detect activation of Factor V under these conditions suggests that <4% of the platelet protease is released by thrombin. Subcellular fractionation of platelets indicates that the platelet protease that activates Factor V is in the soluble fraction.When Factor Va formed by the action of platelet protease is incubated with platelets, peptides with Mr = 105,000, 87,000, and 78,000 bind to the platelet surface. All three radiolabeled peptides are displaced

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Subcellular localization and secretion of factor V from human platelets.

Cited by 70 publications

References 30 publications

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

The assembly of the prothrombinase complex on adherent platelets.

Activation of Coagulation Factor V by a Platelet Protease

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